Signal transduction is a central component of nearly all biological processes. Protein kinases play critical roles in signaling pathways by phosphorylating proteins involved in signal amplification and in executing the cellular response to extrinsic or intrinsic stimuli. Aberrant kinase signaling plays a role in the etiology of nearly all cancers and in hundreds of other diseases. Systematic approaches to the elucidation of the signaling pathways driven by kinases have the potential to illuminate new points of therapeutic intervention. The identification of physiologic kinase substrates is an important component of this endeavor. Currently, there is a need for technologies that can be widely deployed to approach this problem. We are developing a simple, straightforward technique for kinase substrate profiling that we term the reverse in-gel kinase assay. This assay enables the discovery of kinase substrates in whole or pre-fractionated complex biological extracts. If properly developed and validated, this assay has the potential, through the concerted effort of multiple laboratories, to enable rapid identification of potential physiologic substrates of many kinases.